Observation apparatus, observation method, observation system, program, and cell manufacturing method

ABSTRACT

An observation apparatus includes: an area calculation unit that calculates a colony area based on an image in which a cell colony is captured; a cell number calculation unit that calculates, based on the image, the number of cells included in a target colony of which an area is calculated by the area calculation unit; and a density calculation unit that calculates, based on the area of the target colony calculated by the area calculation unit and the number of the cells included in the target colony calculated by the cell number calculation unit, a density of the cells included in the target colony.

RELATED APPLICATION

The present application is a continuation application of U.S. patentapplication Ser. No. 15/319,336 filed Feb. 27, 2017, which in turn is aU.S. national stage application of PCT/JP2014/065916 filed Jun. 16,2014. Each of these prior applications is incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present invention relates to an observation apparatus, anobservation method, an observation system, a program, and a cellmanufacturing method.

BACKGROUND

In general, a technology of evaluating a cell incubation state is a basetechnology in a broad range of fields including an advanced medicalfield such as regenerative medicine and screening of pharmaceuticalproducts. For example, the regenerative medical field includes a processof cell proliferation and cell differentiation in vitro. In the process,in order to manage success and failure of cell differentiation andpresence or absence of cell canceration and infection, it isindispensable to accurately evaluate the cell incubation state. As anexample, an evaluation method of a cancer cell using a transcriptionfactor as a marker is disclosed (refer to Patent Document 1).

A stem cell such as an ES (Embryonic Stem) cell or an iPS (inducedPluripotent Stem) cell can be substantially infinitely proliferatedtheoretically while maintaining differentiation pluripotency that thecell can differentiate into substantially all tissues and therefore hasbeen drawing attention in pharmaceutical development and regenerativemedical applications.

RELATED ART DOCUMENTS Patent Documents

[Patent Document 1] U.S. Pat. No. 7,060,445

SUMMARY OF INVENTION Problems to be Solved by the Invention

When such a stem cell is applied to drug discovery research orregenerative medicine, it is necessary to incubate a stem cell having agood state (the size of a colony is a moderate size, and the density ofa cell that is present in the colony is a moderate density), andtherefore, it is required to accurately determine the maturity degree ofa cell line during incubation. However, in the related art, the maturitydegree of a cell line is determined according to visual observation of aresearcher, and therefore, there is a problem that it is impossible toimprove the determination accuracy of the maturity degree of a cellline.

In view of the foregoing, a problem to be solved by the presentinvention is to provide an observation apparatus, an observation method,an observation system, a program, and a cell manufacturing methodcapable of improving the determination accuracy of the maturity degreeof a cell line.

Means for Solving the Problem

[1] In order to solve the problem, an aspect of the present invention isan observation apparatus including: an area calculation unit thatcalculates a colony area based on an image in which a cell colony iscaptured; a cell number calculation unit that calculates, based on theimage, the number of cells included in a target colony of which an areais calculated by the area calculation unit; and a density calculationunit that calculates, based on the area of the target colony calculatedby the area calculation unit and the number of the cells included in thetarget colony calculated by the cell number calculation unit, a densityof the cells included in the target colony.

[2] Further, in order to solve the problem, an aspect of the presentinvention is an observation system including: an imaging unit thatcaptures an image of a cell during incubation; and an observationapparatus described above.

[3] Further, in order to solve the problem, an aspect of the presentinvention is an observation method including: an area calculation stepof calculating a colony area based on an image in which a cell colony iscaptured; a cell number calculation step of calculating, based on theimage, the number of cells included in a target colony of which an areais calculated according to the area calculation step; and a densitycalculation step of calculating, based on the area of the target colonycalculated according to the area calculation step and the number of thecells included in the target colony calculated according to the cellnumber calculation step, a density of the cells included in the targetcolony.

[4] Further, in order to solve the problem, an aspect of the presentinvention is a program that causes a computer to execute: an areacalculation step of calculating a colony area based on an image in whicha cell colony is captured; a cell number calculation step ofcalculating, based on the image, the number of cells included in atarget colony of which an area is calculated according to the areacalculation step; and a density calculation step of calculating, basedon the area of the target colony calculated according to the areacalculation step and the number of the cells included in the targetcolony calculated according to the cell number calculation step, adensity of the cells included in the target colony.

[5] Further, in order to solve the problem, an aspect of the presentinvention is a cell manufacturing method including: a cell incubationstep of incubating a cell; an area calculation step of imaging a cellcolony incubated in the incubation step and calculating a colony areabased on a captured image of the colony; a cell number calculation stepof calculating, based on the image, the number of cells included in atarget colony of which an area is calculated according to the areacalculation step; and a density calculation step of calculating, basedon the area of the target colony calculated according to the areacalculation step and the number of the cells included in the targetcolony calculated according to the cell number calculation step, adensity of the cells included in the target colony.

Advantage of the Invention

According to the present invention, it is possible to improve thedetermination accuracy of the maturity degree of a cell line.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing an example of a configuration of anobservation apparatus according to an embodiment of the presentinvention.

FIG. 2 is a block diagram showing an example of a configuration of acontrol device included in the observation apparatus of the presentembodiment.

FIG. 3 is a front view of the observation apparatus of the presentembodiment.

FIG. 4 is a plan view of the observation apparatus of the presentembodiment.

FIG. 5 is a graph showing an example of a temporal change of a colonyarea for each cell line of the present embodiment.

FIG. 6 is a graph showing an example of a relationship between a colonyarea and a cell number for each cell line of the present embodiment.

FIG. 7 is a graph showing an example of a relationship between a cellcolony area and the density of the cell included in the colony.

FIG. 8 is a schematic diagram showing an example of an entireobservation image captured by an imaging device of the presentembodiment.

FIG. 9 is a graph showing an example of a relationship between thetemporal change of the colony area and a first calibration curve of thepresent embodiment.

FIG. 10 is a flowchart showing an example of a calibration curveregistration operation according to the observation apparatus of thepresent embodiment.

FIG. 11 is a flowchart showing an example of a cell number estimationoperation according to the observation apparatus of the presentembodiment.

DESCRIPTION OF EMBODIMENTS Embodiment

Hereinafter, an embodiment of the present invention will be describedwith reference to the drawings. First, with reference to FIG. 1 to FIG.4, an outline of a configuration of an incubator (observation apparatus)according to the embodiment of the present invention is described. FIG.1 is a block diagram showing an example of a configuration of anincubator 11 according to the embodiment of the present invention. FIG.2 is a block diagram showing an example of a configuration of a controldevice 41 included in the incubator 11 of the present embodiment. FIG. 3and FIG. 4 are front and plan views of the incubator 11 of the presentembodiment.

The incubator 11 is an apparatus used for incubating a cell andobserving the state of the cell by imaging the incubated cell using amicroscope camera. The incubator 11 has an upper casing 12 and a lowercasing 13. In an assembled state of the incubator 11, the upper casing12 is arranged above the lower casing 13. The internal spaces of theupper casing 12 and the lower casing 13 are separated in the verticaldirection by a base plate 14.

First, an outline of a configuration of the upper casing 12 isdescribed. A constant temperature room 15 in which a cell is incubatedis formed inside the upper casing 12. The constant temperature room 15has a temperature adjustment device 15 a and a humidity adjustmentdevice 15 b, and the inside of the constant temperature room 15 ismaintained to be an environment (for example, an atmosphere at atemperature of 37° C. and a humidity of 90%) suitable for cellincubation (the temperature adjustment device 15 a and the humidityadjustment device 15 b are not shown in FIG. 3 and FIG. 4).

A large door 16, a medium door 17, and a small door 18 are arranged onthe front surface of the constant temperature room 15. The large door 16covers the front surfaces of the upper casing 12 and the lower casing13. The medium door 17 covers the front surface of the upper casing 12and isolates the circumstance of the constant temperature room 15 fromthe external circumstance when the large door is opened. The small door18 is attached to the medium door 17 and is a door used for carrying inand out an incubation container 19 in which a cell is incubated. As theincubation container 19 is carried in and out of the small door 18, itis possible to prevent environmental changes in the constant temperatureroom 15. The airtightness of the large door 16 is maintained by apacking P1, the airtightness of the medium door 17 is maintained by apacking P2, and the airtightness of the small door 18 is maintained by apacking P3.

A stocker 21, an observation unit 22, a container carry device 23, and acarry table 24 are arranged in the constant temperature room 15. Here,the carry table 24 is arranged in front of the small door 18 and is usedto carry in and out the incubation container 19 from the small door 18.

The stocker 21 is arranged on the left side of the constant temperatureroom 15 when seen from the front surface (lower side of FIG. 4) of theupper casing 12. The stocker 21 has a plurality of shelves, and eachshelf of the stocker 21 can store a plurality of incubation containers19. A cell as a target of incubation together with a culture medium isstored in each of the incubation containers 19. The stocker 21 is notessential.

The observation unit 22 is arranged on the right side of the constanttemperature room 15 when seen from the front surface of the upper casing12. A time lapse observation of a cell in the incubation container 19can be performed using the observation unit 22.

The observation unit 22 is arranged to be fitted in an opening part ofthe base plate 14 of the upper casing 12. The observation unit 22 has asample table 31, a stand arm 32 that projects above the sample table 31and on which an illumination light source is arranged, and a main bodypart 33 that includes an observation system and an imaging device 34.The sample table 31 and the stand arm 32 are arranged in the constanttemperature room 15. On the other hand, the main body part 33 is storedin the lower casing 13.

The sample table 31 is formed of a translucent material, and theincubation container 19 can be arranged on the sample table 31. Thesample table 31 is configured to be movable in the horizontal directionand can adjust the position of the incubation container 19 arranged onthe upper surface of the sample table 31. The stand arm 32 includes aLED light source 39. The imaging device 34 images, via a microscopeoptical system, a cell in the incubation container 19 that isilluminated according to transmission illumination from the upper sideof the sample table 31 by the stand arm 32 and thereby can obtain amicroscope image of the cell.

The container carry device 23 is arranged at the center of the constanttemperature room 15 when seen from the front surface of the upper casing12. The container carry device 23 exchanges the incubation container 19among the stocker 21, the sample table 31 of the observation unit 22,and the carry table 24. When the stocker 21 is not provided as describedabove, the container carry device 23 is also unnecessary.

As shown in FIG. 4, the container carry device 23 has a vertical robot38 having a multijoint arm, a rotation stage 35, a mini stage 36, and anarm unit 37. The rotation stage 35 is attached rotatably by 180° in thehorizontal direction to the front end part of the vertical robot 38 viaa rotation shaft 35 a. Therefore, the rotation stage 35 can cause thearm unit 37 to face each of the stocker 21, the sample table 31, and thecarry table 24.

The mini stage 36 is attached slidably in the horizontal direction withrespect to the rotation stage 35. The arm unit 37 that grips theincubation container 19 is attached to the mini stage 36.

Next, an outline of a configuration of the lower casing 13 is described.The main body part 33 of the observation unit 22 and the control device41 of the incubator 11 are stored inside the lower casing 13.

The control device 41 is connected to each of the temperature adjustmentdevice 15 a, the humidity adjustment device 15 b, the observation unit22, and the container carry device 23. The control device 41 controlsoverall the units of the incubator 11 in accordance with a predeterminedprogram.

As an example, the control device 41 controls each of the temperatureadjustment device 15 a and the humidity adjustment device 15 b andmaintains the inside of the constant temperature room 15 to be apredetermined environmental condition. The control device 41 controls,based on a predetermined observation schedule, the observation unit 22and the container carry device 23 and automatically performs anobservation sequence of the incubation container 19. The control device41 performs, based on the image acquired in the observation sequence, anincubation state evaluation process in which the incubation state of thecell is evaluated.

[Calibration Curve (First Calibration Curve) Indicating Temporal Changeof Cell Colony Area]

With reference to FIG. 5, a temporal change of a cell colony area isdescribed.

FIG. 5 is a graph showing an example of a temporal change of a colonyarea for each cell line. The cells proliferate as time elapses, andtherefore, the area of a colony including these cells (hereinafter,simply referred to also as a colony area) increases according to theelapse of time. The temporal change of the colony area differs for eachcell line. As an example, the temporal change of the colony area of acell line A is indicated by a calibration curve L1A in FIG. 5. Thetemporal change of the colony area of a cell line B is indicated by acalibration curve L1B in FIG. 5. The calibration curve L1A and thecalibration curve L1B are collectively referred to as a firstcalibration curve L1. When the cell line is specified, it is possible toestimate the change of the colony area of a cell line in accordance withthe elapse of time based on a predetermined relation shown in FIG. 5,that is, the first calibration curve L1.

By observing the temporal change of the colony area, it is possible todetermine whether or not the colony is proliferating normally. That is,when the temporal change amount of the colony area of one colony islarger than a standard temporal change amount of the colony area (forexample, when the colony area abnormally increases), there is apossibility that the cell included in this colony differentiates. Thatis, in a colony of which the colony area abnormally increases, it can bedeemed that the cell differentiates, and it is possible to determinethat the colony is an abnormal colony. On the other hand, when thetemporal change amount of the colony area of one colony is smaller thanthe standard temporal change amount of the colony area (for example,when the colony area does not temporally change), there is a possibilitythat the cells included in this colony are dead. That is, in a colony ofwhich the colony area does not temporally change, it can be deemed thatthe cells are dead, and it is possible to determine that the colony isan abnormal colony. The first calibration curve L1 is registered(stored) in advance for each cell line in the incubator 11 of thepresent embodiment, and thereby, the incubator 11 determines whether ornot the colony is proliferating normally.

[Calibration Curve (Second Calibration Curve) Indicating RelationshipBetween Cell Colony Area and Cell Number Included in this Colony]

With reference to FIG. 6, the relationship between a cell colony areaand a cell number is described. The unit of the horizontal axis in FIG.6 is μm² (micro·square meter), and the unit of the vertical axis isnumber.

FIG. 6 is a graph showing an example of a relationship between a colonyarea of one colony for each cell line (hereinafter, colony area) and acell number in one colony (hereinafter, colony cell number). The userselects a colony suitable for a calibration curve in appearance as acolony which is a target when a calibration curve is prepared. The pointof selection is, for example, that a colony which is not adhered toanother colony and which is present independently as an individualcolony is preferable. The colony size is visually determined, and a widevariety of colonies from a colony having a small colony size to a colonyhaving a large colony size are selected. Such selection may be performedvisually according to user's determination, or automatic determinationalso can be made according to an image analysis by storing the selectioncondition in advance.

There is a predetermined relationship between a cell colony area and acell number included in the colony. The predetermined relationshipdiffers for each cell line. As an example, the relationship between thecolony area of a cell line A and the cell number included in the colonyis indicated by a calibration curve L2A in FIG. 6. The relationshipbetween the colony area of a cell line B and the cell number included inthe colony is indicated by a calibration curve L2B in FIG. 6. Thecalibration curve L2A and the calibration curve L2B are collectivelyreferred to as a second calibration curve L2. When the cell line isspecified, it is possible to estimate the cell number included in thecolony from the colony area of the cell line based on the secondcalibration curve L2 shown in FIG. 6. The second calibration curve L2 isregistered (stored) in advance for each cell line in the incubator 11 ofthe present embodiment, and thereby, the incubator 11 estimates(calculates) the cell number included in the colony from the colonyimage. Specifically, with respect to a certain cell line, therelationship between the cell number and the colony area is measuredbased on a phase difference image and a fluorescence image which is animage of a fluorescently-stained cell line. The measured relationshipbetween the cell number and the colony area is stored in advance as thesecond calibration curve L2.

An image used for measuring the cell number is, for example, an image ofa cell in a colony captured in a state where the cell is fluorescentlystained. Then, with respect to the acquired image, the cell number ofcells indicating a predetermined brightness value is measured. At thistime, it is possible to apply a smoothing process with respect to thebrightness value data obtained from the image, and based on the data, itis possible to specify the cell indicating the predetermined brightnessvalue as a cell to be counted. By controlling the process level when thesmoothing process is performed, it is possible to adjust the sensitivityfor specifying the cell.

[Calibration Curve (Third Calibration Curve) Indicating Relationshipbetween Cell Colony Area and Cell Density Included in this Colony]

With reference to FIG. 7, the relationship between a cell colony areaand the density of the cell included in the colony is described.

FIG. 7 is a graph showing an example of a relationship between a cellcolony area and the density of the cell included in the colony. Thedensity of the cell included in the colony increases in response to thecell maturity in accordance with the elapse of time. When the cellmaturity reaches a certain maturity, the change with the elapse of timeof the density of the cell included in the colony is decreased. Thismeans that in accordance with the increase of the colony area, the cellnumber in the colony is increased, but the change of the area perindividual one cell in the colony is decreased.

Accordingly, by observing the density of the cell included in thecolony, it is possible to determine the cell maturity.

Specifically, the density of the cell included in the colony isrepresented by a relationship between the colony area and the area perone cell included in the colony. The colony proliferates over time, andtherefore, the colony area increases. When the maturity of the cellincluded in the colony is increased, the inside of the colony becomes apacked state. That is, when the cell included in the colony is matured,the density of the cell inside the colony is increased. When the cellfurther proliferates in a state where the inside of the colony ispacked, the density of the cell reaches an upper limit to suppress theincrease of the density, and the colony area is increased. Accordingly,by observing the temporal change of the density of the cell inside thecolony, it is possible to determine the maturity of the cell included inthe colony.

The density of the cell included in the colony differs for each cellline. As an example, the relationship between the colony area of a cellline A and the area per one cell included in the colony is indicated bya calibration curve L3A in FIG. 7. The relationship between the colonyarea of a cell line B and the area per one cell included in the colonyis indicated by a calibration curve L3B in FIG. 7. The calibration curveL3A and the calibration curve L3B are collectively referred to as athird calibration curve L3. When the cell line is specified, it ispossible to determine the maturity of the cell based on the thirdcalibration curve L3 shown in FIG. 7. Specifically, with respect to thecell line A, when the area per one cell included in the colony reaches athreshold value ThA of the calibration curve L3A shown in FIG. 7, it isdetermined that the cell line A is matured. With respect to the cellline B, when the area per one cell included in the colony reaches athreshold value ThB of the calibration curve L3B shown in FIG. 7, it isdetermined that the cell line B is matured.

That is, the third calibration curve L3 is registered (stored) inadvance for each cell line in the incubator 11 of the presentembodiment, and thereby, the incubator 11 determines the cell maturityfrom the colony image. Specifically, when a calibration curve isprepared, with respect to a certain cell line, the relationship betweenthe colony area and the density of the cell included in the colony ismeasured based on a phase difference image and a fluorescence imagewhich is an image of a fluorescently-stained cell line. The measuredrelationship between the colony area and the density of the cellincluded in the colony is stored in advance as the third calibrationcurve L3.

With reference back to FIG. 1, the configuration of the control device41 is described. The control device 41 has a control unit 42, a storageunit 43, and an input unit 44.

The storage unit 43 is formed of a hard disk, a non-volatile storagemedium such as a flash memory, a volatile storage medium such as a DRAMand a SRAM, or the like. Management data regarding each incubationcontainer 19 stored in the stocker 21, data of an entire observationimage captured by an imaging device, and data of a microscope image arestored in the storage unit 43. A program executed by the control unit 42is stored in the storage unit 43. A variety of calculation results bythe control unit 42 are temporarily stored in the storage unit 43.

The management data described above includes (a) index data indicatingan individual incubation container 19, (b) a storage position of theincubation container 19 in the stocker 21, (c) the type and shape (wellplate, dish, flask, or the like) of the incubation container 19, (d) thetype (information by which a cell line is identified) of the cellincubated in the incubation container 19, (e) an observation schedule ofthe incubation container 19, (f) an imaging condition (a magnificationof an objective lens, an observation point in the container, or thelike) during a time lapse observation, and the like. With respect to theincubation container 19 in which cells can be incubated simultaneouslyat a plurality of small containers such as a well plate, each of themanagement data is generated for each of the plurality of smallcontainers.

In the present example, different types of cell lines are observed asthe cell line to be observed. In this case, information by which thecell line is identified is required. However, when the cell line whichis observed is one cell line, and it is unnecessary to identify the cellline, the cell line identification information is not essential. Evenwhen the cell line which is observed is one cell line, informationindicating the cell line may be input.

The colony area and calibration curve information indicating therelationship between the colony area and the number of cells included inthe colony are associated with each other and stored in the storage unit43.

When different types of cell lines are observed, cell line informationby which a cell line of a cell is identified is stored in the storageunit 43 and is preferably associated with each of information to bestored. Feature amount information indicating a feature amount of thecolony area is stored and is preferably associated with each ofinformation to be stored.

The input unit 44 includes an input device such as a keyboard and amouse. A variety of information such as cell line information are inputto the input unit 44 according to the operation of the user.

Next, with reference to FIG. 2, the configuration of the control unit 42is described. The control unit 42 includes an image read unit 4203, anarea calculation unit 4211, a write control unit 4221, a cell numbercalculation unit 4222, and a density calculation unit 4223.

In FIG. 2, a case in which it is determined whether the colony is goodor bad based on the first calibration curve L1 is supposed, and aconfiguration in which a quality determination unit 4224 is included isshown. In FIG. 2, a case in which the cell maturity is determined basedon the third calibration curve L3 is supposed, and a configuration inwhich a maturity determination unit 4225 is included is shown. In theincubator 11 of the present embodiment, the quality determination unit4224 and the maturity determination unit 4225 are not essential.

The control unit 42 is, for example, a processor that performs a varietyof calculation processes of the control device 41. The control unit 42may function as each of the image read unit 4203, the area calculationunit 4211, the quality determination unit 4224, the maturitydetermination unit 4225, the write control unit 4221, and the cellnumber calculation unit 4222 by executing a program.

The write control unit 4221 controls writing, in the storage unit 43, ofinformation output by each unit of the control device 41.

The image read unit 4203 reads the image data of the microscope image orthe entire observation image captured by the imaging device 34 andsupplies the read image data to each unit of the control device 41. Theimage read unit 4203 reads the image data of the microscope image or theentire observation image stored in the storage unit 43 and supplies theread image data to each unit of the control device 41.

In two cases which are a case in which the first calibration curve L1 isstored in the storage unit 43 and a case in which it is determinedwhether the colony is good or bad based on the first calibration curveL1 and the colony image captured by the imaging device 34, the areacalculation unit 4211 calculates the colony area. Here, first, the casein which the first calibration curve L1 is stored in the storage unit 43is described, and then, the case in which it is determined whether thecolony is good or bad based on the first calibration curve L1 isdescribed.

The area calculation unit 4211 calculates the colony area and causes thestorage unit 43 via the write control unit 4221 to store, as calibrationcurve information, the first calibration curve L1 in which thecalculated colony area and the number of cells included in the colonyare associated with each other. That is, the area calculation unit 4211causes the storage unit 43 to store the first calibration curve L1. Incase of a plurality of cell lines, the storage unit 43 is caused tostore cell line information (cell line ID) via the input unit 44, andthe storage unit 43 is caused to store calibration curve information foreach cell line.

In case of a plurality of types of cell lines, it is required to inputinformation (cell line ID) indicating a cell line type. With respect tothe input of information indicating a cell line type, the userunderstanding the types of observed cell lines may input theinformation. Further, by using a technology in which the cell line typeis automatically determined using a matching technique or the like ofidentifying a cell according to the morphology, brightness, and the likeof the observed cell, the information indicating a cell line type can bealso automatically generated to be input.

In the present example, a case in which the user inputs the information(cell line ID) indicating the cell line via the input unit 44 isdescribed.

The area calculation unit 4211 calculates the colony area based on theimage in which the cell colony is captured. A specific example of theimage of which the colony area is calculated by the area calculationunit 4211 is described with reference to FIG. 8.

FIG. 8 is a schematic diagram showing an example of an entireobservation image captured by the imaging device 34 of the presentembodiment. Among the entire observation image shown in FIG. 8, anentire observation image PIC01 is an image (frame 1) of a colonydetection result after one day elapses from the start of incubation.Entire observation image PIC02 to entire observation image PIC05 (frame1 to frame 5) are images of colony detection results after two to fivedays elapse from the start of incubation.

The entire observation image PIC01 includes an image of a first colonyCO1 and an image of a second colony CO2 after one day elapses from thestart of incubation. The entire observation image PIC02 includes animage of the first colony CO1 and an image of the second colony CO2after two days elapse from the start of incubation. As shown in FIG. 8,the first colony CO1 after two days elapse from the start of incubationproliferates compared to the first colony CO1 after one day elapses fromthe start of incubation, and the area is increased. As shown in FIG. 8,the second colony CO2 after two days elapse from the start of incubationproliferates compared to the second colony CO2 after one day elapsesfrom the start of incubation, and the area is increased.

The entire observation image PIC03 includes an image of the first colonyCO1, an image of the second colony CO2, and an image of a third colonyCO3 after three days elapse from the start of incubation. As shown inFIG. 8, the first colony CO1 after three days elapse from the start ofincubation proliferates compared to the first colony CO1 after two dayselapse from the start of incubation, and the area is increased. As shownin FIG. 8, the second colony CO2 after three days elapse from the startof incubation proliferates compared to the second colony CO2 after twodays elapse from the start of incubation, and the area is increased.

The temporal change of the colony area is described by sorting theentire observation image PIC01 to the entire observation image PIC03 ina time series and comparing the images. In this case, the area of thefirst colony CO1 monotonously increases in the entire observation imagePIC01 to the entire observation image PIC03. On the other hand, withrespect to the area of the second colony CO2, the increase amount in theentire observation image PIC01 to the entire observation image PIC02greatly differs from the increase amount in the entire observation imagePIC02 to the entire observation image PIC03. That is, the increaseamount of the area of the second colony CO2 in the entire observationimage PIC02 to the entire observation image PIC03 is larger than theincrease amount of the area of the second colony CO2 in the entireobservation image PIC01 to the entire observation image PIC02. That is,the area of the second colony CO2 drastically increases after three dayselapse from the start of incubation. This indicates that there is apossibility that the second colony CO2 differentiates after three dayselapse from the start of incubation and abnormally proliferates.

With reference to FIG. 9, the relationship between the temporal changeof the colony area and the first calibration curve is described.

FIG. 9 is a graph showing an example of the relationship between thetemporal change of the colony area and the first calibration curve ofthe present embodiment. In FIG. 9, the horizontal axis represents time,and the vertical axis represents colony area. FIG. 9(A) shows the areaof the colony included in the entire observation image PIC01 obtained byacquiring the image of the colony. That is, FIG. 9(A) shows the area ofthe first colony CO1 and the area of the second colony CO2 after one dayelapses from the start of incubation. As shown in FIG. 9(A), both anarea CO11 of the first colony CO1 and an area CO21 of the second colonyCO2 are plotted on the first calibration curve L1. That is, after oneday elapses from the start of incubation, both the area CO11 of thefirst colony CO1 and the area CO21 of the second colony CO2 show anormal value.

Each of FIG. 9(B) to (E) shows the area of the colony included in eachof the entire observation image PIC02 to the entire observation imagePIC05 obtained by acquiring the image of the colony. FIG. 9(B) shows anarea CO12 of the first colony CO1 and an area CO22 of the second colonyCO2 after two days elapse from the start of incubation. As shown in FIG.9(B), both the area CO12 of the first colony CO1 and the area CO22 ofthe second colony CO2 are plotted on the first calibration curve L1.That is, after two days elapse from the start of incubation, both thearea CO12 of the first colony CO1 and the area CO22 of the second colonyCO2 show a normal value.

FIG. 9(C) shows an area CO13 of the first colony CO1 and an area CO23 ofthe second colony CO2 after three days elapse from the start ofincubation. As shown in FIG. 9(C), the area CO13 of the first colony CO1is plotted on the first calibration curve L1. On the other hand, thearea CO23 of the second colony CO2 is not plotted on the firstcalibration curve L1. That is, after three days elapse from the start ofincubation, the area CO12 of the first colony CO1 shows a normal value,and the area CO23 of the second colony CO2 shows an abnormal value. Thisindicates that there is a possibility that the second colony CO2differentiates after three days elapse from the start of incubation andabnormally proliferates. That is, it is possible to determine whetherthe colony area is a normal value or is an abnormal value based onwhether or not the colony area is present on the first calibration curveL1. In this example, since the second colony CO2 is in a state where thecolony area is greater than that of a normal colony, and there is apossibility that the second colony CO2 differentiates after three dayselapse from the start of incubation and abnormally proliferates, thesubsequent incubation of the second colony CO2 is stopped (for example,remove the second colony CO2 from the culture medium).

In the present embodiment, the quality determination unit 4224determines whether the colony is good or bad. That is, the qualitydetermination unit 4224 determines whether the colony is good or badbased on the change of the colony area according to the elapse of timecalculated by the area calculation unit 4211. Specifically, the qualitydetermination unit 4224 determines whether the colony is good or badbased on the colony area calculated by the area calculation unit 4211and the first calibration curve L1 stored in the storage unit 43.

With reference back to FIG. 8, after three days elapse from the start ofincubation, the third colony CO3 that has not been present beforearises. This is because an iPS cell is different from an ordinary celland the timing of adhesion is non-uniform. Specifically, the firstcolony CO1 and the second colony CO2 adhere and proliferate after oneday elapses from the start of incubation. On the other hand, the thirdcolony CO3 does not adhere until two days elapse from the start ofincubation and adhere after three days elapse from the start ofincubation to start proliferating. In this way, the imaging device 34generates an entire observation image by imaging multiple times inaccordance with the elapse of time from the start of incubation.

An entire observation image PIC04 (frame 4) includes an image of thefirst colony CO1, an image of the second colony CO2, and an image of thethird colony CO3 after four days elapse from the start of incubation. Asshown in FIG. 8, the first colony CO1 after four days elapse from thestart of incubation proliferates compared to the first colony CO1 afterthree days elapse from the start of incubation, and the area isincreased. As shown in FIG. 8, the second colony CO2 after four dayselapse from the start of incubation proliferates compared to the secondcolony CO2 after two days elapse from the start of incubation, and thearea is increased.

That is, the imaging device 34 captures a time lapse image from thestart of incubation. Thereby, the incubator 11 of the present embodimentcan observe the state of colony with good accuracy even with respect toa cell of which the timing of adhesion is non-uniform such as an iPScell.

With reference back to FIG. 2, the area calculation unit 4211 acquiresthe entire observation image captured by the imaging device 34 from theimage read unit 4203. The area calculation unit 4211 generates a colonydetection result image (the entire observation image PIC01 to the entireobservation image PIC05) shown in FIG. 8 based on the entire observationimage which was acquired.

The area calculation unit 4211 calculates the colony area of a cellbased on the generated colony detection result image. Specifically, thearea calculation unit 4211 masks a colony part using an object detectionalgorithm according to a known learning function, determines the maskedpart (region surrounded by a curve in the colony detection result imageof FIG. 8) as a region where the colony is present, and calculates thecolony area from the masked region. The calculation method of the colonyarea is not limited thereto.

When the user inputs information (cell line ID) indicating a cell linevia the input unit 44, the cell number calculation unit 4222 searchescalibration curve information stored by the storage unit 43 using thecell line ID as a search key and acquires the second calibration curveL2 (relationship between the colony area and the cell number) which thecell line ID matches based on the search result. The cell numbercalculation unit 4222 calculates the cell number based on the colonyarea calculated by the area calculation unit 4211 using the acquiredsecond calibration curve L2. That is, the cell number calculation unit4222 calculates the number of cells included in the target colony ofwhich the area is calculated by the area calculation unit 4211 based onthe image captured by the imaging device 34.

The density calculation unit 4223 calculates the density of cellsincluded in the colony based on the colony area calculated by the areacalculation unit 4211 and the number of cells included in the colonycalculated by the cell number calculation unit 4222. Specifically, thedensity calculation unit 4223 calculates the area per one cell includedin the colony based on the colony area calculated by the areacalculation unit 4211 and the cell number calculated by the cell numbercalculation unit 4222.

The maturity determination unit 4225 determines the maturity of the cellincluded in the colony based on the density of the cell calculated bythe density calculation unit 4223. Specifically, with respect to thecell line A, when the area per one cell included in the colony reachesthe threshold value ThA of the calibration curve L3A shown in FIG. 8,the maturity determination unit 4225 determines that the cell line A ismatured. With respect to the cell line B, when the area per one cellincluded in the colony reaches the threshold value ThB of thecalibration curve L3B shown in FIG. 8, the maturity determination unit4225 determines that the cell line B is matured.

[Operation of Incubator (Observation Apparatus)]

Next, an example of an operation of the incubator 11 is described. Theincubator 11 estimates the number of cells included in the colony fromthe cell colony area based on the registered calibration curve tothereby calculate the number of cells. Here, the calibration curve isinformation indicating the relationship between the cell colony area andthe number of cells included in the colony. First, an operation in whichthe calibration curve is registered is described, and then, an operationin which the number of cells is calculated based on the registeredcalibration curve is described.

[Operation of Calibration Curve Registration]

First, an operation of calibration curve registration is described withreference to FIG. 10.

FIG. 10 is a flowchart showing an example of an operation of calibrationcurve registration by the incubator 11 (observation apparatus) of thepresent embodiment. The incubator 11 stores a calibration curve (firstcalibration curve L1, second calibration curve L2, and third calibrationcurve L3) for each cell line.

As an example, based on the image data described above, the incubator 11detects a colony image in the image data and calculates the area of thecolony image. The incubator 11 associates the calculated colony areawith the elapsed time from the start of incubation for each cell line tothereby register a first calibration curve L1 with respect to a certaincell line.

The incubator 11 associates the calculated colony area with the numberof cells in the colony counted according to a fluorescently-stainedobservation image for each cell line to thereby register a secondcalibration curve L2 with respect to a certain cell line as calibrationcurve information.

The incubator 11 associates the calculated colony area with an area perone cell in the colony for each cell line to thereby register a thirdcalibration curve L3 with respect to a certain cell line as calibrationcurve information.

Before the operation start of calibration curve registration, thecontrol unit 42 preliminarily accepts a command of a calibration curveregistration operation input via the input unit 44 by the user. Thecommand of the calibration curve registration operation includesinformation (cell line ID) indicating a cell line of which thecalibration curve is to be registered (when the observed cell line isone cell line, the input of information (cell line ID) indicating a cellline of which the calibration curve is to be registered is notessential). The storage unit 43 preliminarily stores observation starttimes as an observation schedule of management data such that anobservation is started after each day elapses from the elapse of one daysince the start of incubation until the elapse of five days. When themethod by which the user counts the number of cells in the colony is amethod in which a cell in the incubation container 19 is destructed andis observed such as a counting method in which cells arefluorescently-stained and are counted according to a fluorescentobservation, incubation containers 19 are prepared corresponding to thenumber of observation times. For example, when the observation isperformed after the elapse of each of one day, two days, and three daysfrom the start of incubation, at least three incubation containers 19are stored in the stocker 21 of the constant temperature room 15. Thesame cell line is incubated in each of the incubation containers 19. Byobserving the incubation containers 19 one by one per one day, the cellline after the elapse of one day from the start of incubation to thecell line after the elapse of five days from the start of incubation areobserved. The specific operation of preparation of the calibration curveperformed by the incubator 11, that is, calculation of the colony areaand registration of the calibration curve is described below. In thepresent embodiment, the following operation is performed by the controlunit 42 included in the incubator 11; however, the following operationmay be performed by a control unit externally provided on the incubator11.

Step S101: The control unit 42 determines whether or not the observationstart time of the incubation container 19 has come by comparing theobservation schedule of the management data of the storage unit 43 andthe current date and time. When it is the observation start time (YES),the control unit 42 forwards the process to Step S102. On the otherhand, when it is not the observation time (NO), the control unit 42waits until the next observation schedule time.

Step S102: The control unit 42 commands the container carry device 23 tocarry the incubation container 19 corresponding to the observationschedule. Then, the container carry device 23 carries out the commandedincubation container 19 from the stocker 21 and places the incubationcontainer 19 on the sample table 31 of the observation unit 22. At thetiming when the incubation container 19 is placed on the sample table31, the entire observation image of the incubation container 19 iscaptured by a bird view camera (not shown) embedded in the stand arm 32.Thereby, the image of the incubation container 19 including a colonyimage is captured. As described above, the stocker 21 is not essential.When there is no stocker 21, the step regarding the container carry isunnecessary.

Step S103: The image read unit 4203 of the control unit 42 stores theentire observation image captured in Step S102. The area calculationunit 4211 of the control unit 42 detects a colony image from the storedentire observation image and calculates the sum of areas of colony cellsin the incubation container 19 or the sum of areas of colonies as thecolony area.

When the calibration curve is prepared, the image of the incubationcontainer including the colony image may be acquired, the cell number ofeach colony may be counted (or the cell number of each colony may besummed) from the fluorescent observation image, and the colony area maybe calculated from the phase difference image. Alternatively, the totalnumber of cells in each colony may be counted by a hemocytometer, andthe colony area may be calculated from the phase difference image.

Step S104: The control unit 42 commands the container carry device 23 tocarry the incubation container 19 to the small door 18 after theobservation schedule is finished. Then, the container carry device 23carries the commanded incubation container 19 to the position of thesmall door 18 from the sample table 31 of the observation unit 22.

Step S105: The user opens the small door 18 and takes out the incubationcontainer 19. The user counts the number of cells according to a knownmethod with respect to the incubation container 19 which is taken out.For example, the user counts the number of cells according tofluorescence stain. The user inputs the counted number of cells to theinput unit 44. Here, the user calculates the cell density in the colonyand inputs the calculated density to the input unit 44.

Step S106: The write control unit 4221 of the control unit 42 associatesthe cell number in the colony and the cell density in the colony inputto the input unit 44 with the colony area calculated in Step S103 andthe information (cell line ID) indicating the cell line to be stored inthe storage unit 43. Thereby, the colony area, the cell number, and thecell line ID are associated with one another and stored in the storageunit 43 as calibration curve information (in a case of observation ofone cell line, the association with the cell line ID is not essential).Then, the control unit 42 finishes the observation sequence and causesthe process to return to Step S101.

The calibration curve information is stored in the storage unit 43 byrepeating Step S101 to Step S106 in this way. By repeating Step S101 toStep S106 with respect to a plurality of cell lines, the calibrationcurve information with respect to each of the plurality of cell lines isstored in the storage unit 43. Specifically, by repeating Step S101 toStep S106 with respect to each of the cell line A and the cell line B,each of the calibration curve information of the cell line A and thecalibration curve information of the cell line B is stored in thestorage unit 43.

[Operation of Cell Number Estimation and Cell Quality Determination]

Next, with reference to FIG. 11, an example of the cell numberestimation operation of the incubator 11 is described.

FIG. 11 is a flowchart showing an example of a cell number estimationoperation according to the incubator 11 (observation apparatus) of thepresent embodiment. In the present example, data (referred to ascontrast data) regarding the number of cells against the change of thecolony area of a specific cell line which is to be an observation targetis acquired and is stored in advance. From the data, the determinationof the quality of the observed cell is in an unknown state.

The incubator 11 performs a time lapse observation of the incubationcontainer 19 carried in the constant temperature room 15 in accordancewith the registered observation schedule. A plurality of specific typesof cell lines are incubated in the incubation container 19. For example,the cell line A is incubated in an incubation container 19A of theincubation containers 19. The cell line B is incubated in an incubationcontainer 19B of the incubation containers 19. In accordance with theobservation schedule, the incubator 11 sequentially carries theincubation container 19A and the incubation container 19B to thevertical robot 38 and the observation unit 22 and captures an entireimage (entire observation image) of the incubation container 19 and amicroscope image in which part of the incubation container 19 ismagnified. According to the registration sequence of the calibrationcurve information described above, the calibration curve information ofthe cell line A and the calibration curve information of the cell line Bare stored in the storage unit 43 in advance. The operation of cellnumber estimation in the time lapse observation of the incubator 11 isdescribed.

Step S201: The control unit 42 determines whether or not the observationstart time of the incubation container 19 has come by comparing theobservation schedule of the management data of the storage unit 43 andthe current date and time. When it is the observation start time (YES),the control unit 42 forwards the process to Step S202. On the otherhand, when it is not the observation time of the incubation container 19(NO), the control unit 42 waits until the next observation scheduletime.

Step S202: The control unit 42 commands the container carry device 23 tocarry the incubation container 19 corresponding to the observationschedule. Then, the container carry device 23 carries out the commandedincubation container 19 from the stocker 21 and places the incubationcontainer 19 on the sample table 31 of the observation unit 22. At thetiming when the incubation container 19 is placed on the sample table31, the entire observation image of the incubation container 19 iscaptured by a bird view camera (not shown) embedded in the stand arm 32.

In the present example, the observation apparatus shown in FIGS. 1, 3,and 4 is an apparatus including a constant temperature room in which acell to be observed is incubated. Accordingly, the cell imaged in StepS202 is a cell incubated in a constant temperature room of theobservation apparatus. Accordingly, the present step can be also startedfrom the step of incubating a cell. The apparatus may not be anapparatus in which the constant temperature room is included in theobservation apparatus like the present example. The apparatus may be anapparatus in which the constant temperature room for incubating a cellis separated from the observation apparatus.

Step S203: The cell number calculation unit 4222 that calculates thecell number in the colony acquires the information (cell line ID)indicating the cell line from the management data stored in the storageunit 43.

Step S204: The cell number calculation unit 4222 searches calibrationcurve information stored by the storage unit 43 using the acquired cellline ID as a search key and acquires calibration curve information whichthe cell line ID matches.

Step S205: The image read unit 4203 acquires an image captured in StepS202. The image includes an image of a colony.

Step S206: The area calculation unit 4211 calculates, based on the imageacquired in Step S205, the area of the colony included in the image.

Step S207: The cell number calculation unit 4222 estimates (calculates)the number of cells based on the area of the colony calculated in StepS206 and a second calibration curve L2 of the calibration curveinformation acquired in Step S204.

The area calculation unit 4211 calculates the area of the recognizedcolony in the acquired image.

This step may be performed when a state in which observation can be madeis realized after the incubation container is carried to the observationunit (S202). In this case, after the colony area is calculated, theinformation indicating a cell line is acquired.

Step S208: The density calculation unit 4223 calculates a cell densitybased on the area of the colony calculated in Step S206 and the numberof cells calculated in Step S207.

Step S209: When the quality determination unit 4224 is included, thequality determination unit 4224 determines whether the colony is good orbad based on the colony area calculated from the acquired image and afirst calibration curve L1 of the calibration curve information acquiredin Step S204. When the quality determination unit 4224 is not included,the user determines whether the colony is good or bad based on thecalculated colony area and the first calibration curve L1 of thecalibration curve information acquired in Step S204.

When the maturity determination unit 4225 is included, the maturitydetermination unit 4225 determines the cell maturity based on the colonyarea calculated in Step S206, the cell density calculated in Step S208,and a third calibration curve L3 of the calibration curve informationacquired in Step S204 by using the acquired image. When the maturitydetermination unit 4225 is not included, the user determines the cellmaturity based on the colony area calculated in Step S206, the celldensity calculated in Step S208, and the third calibration curve L3 ofthe calibration curve information acquired in Step S204.

Step S210: The control unit 42 commands the container carry device 23 tocarry the incubation container 19 after the observation schedule isfinished. Then, the container carry device 23 carries the commandedincubation container 19 from the sample table 31 of the observation unit22 to a predetermined storage position of the stocker 21. Then, thecontrol unit 42 finishes the observation sequence and causes the processto return to S201.

In addition, after it can be understood whether or not a cell is in agood state as a result of determination in Step S209, it is possible totake out a cell which is determined that the cell is in a good statefrom the observation apparatus and, for example, supply the cell as acell used for drug discovery research or regenerative medicine.

By repeating Step S201 to Step S210 for each cell line in this way, theestimation result of the number of cells in each observation schedule isstored in the storage unit 43. Further, it is possible to determinewhether the cell is good or bad based on the estimation result of thenumber of cells. Further, it is possible to supply only the selectedgood cell to a research institute or the like.

As described above, the incubator 11 (observation apparatus) of thepresent embodiment includes the cell number calculation unit 4222 thatcalculates, based on the calibration curve information and the colonyarea on the basis of the non-invasively obtained image, the number ofcells included in the colony. Thereby, the incubator 11 can calculatethe number of cells according to a non-invasive method and can improvethe accuracy of the calculated number of cells. Further, an imageobtained by a non-invasive observation (for example, phase differenceobservation) is used for the maturity determination or qualitydetermination, and therefore, the determined cell can be subsequentlyused without obstacle for drug discovery research or regenerativemedicine as a subsequent process.

The control device 41 may treat a plurality of microscope imagescaptured during the same period of observation time of a plurality ofpoints (for example, five points of observation or the entire incubationcontainer 19) of the same incubation container 19 as an image of onetime of the time lapse observation.

The embodiment is described using an example in which the areacalculation unit 4211 detects the colony image based on the entireobservation image; however, the embodiment is not limited thereto. Thearea calculation unit 4211 may detect the colony image by imageprocessing of a phase difference microscope image.

A program for executing each process of the incubator 11 (observationapparatus) according to the embodiments of the invention may be recordedin a computer-readable recording medium, and the program recorded in therecording medium may be read into and executed on a computer system tothereby perform a variety of processes described above.

It is assumed that the “computer system” used herein includes an OS orhardware such as peripherals. It is also assumed that the term “computersystem” includes a homepage provision environment (or a displayenvironment) when a WWW system is used. The term “computer-readablerecording medium” refers to a recordable non-volatile memory such as aflexible disk, a magneto-optical disk, a ROM, or a flash memory, aportable medium such as a CD-ROM, or a storage device such as a harddisk embedded in the computer system.

It is also assumed that the term “computer-readable recording medium”includes a medium which holds a program for a given time such as avolatile memory (for example, a DRAM (Dynamic Random Access Memory)) inthe computer system which becomes a server or a client when a program istransmitted through a network such as the Internet or a communicationline such as a telephone line. The program may be transmitted from thecomputer system which stores the program in the storage device or thelike to other computer systems through a transmission medium or throughtransmission waves in the transmission medium. The term “transmissionmedium” which transmits the program refers to a medium which has afunction of transmitting information, for example, a network(communication network) such as the Internet or a communication linesuch as a telephone line. The program may be a program which can realizepart of the above-described functions. The program may be a so-calleddifferential file (differential program) which can realize theabove-described functions by a combination with a program alreadyrecorded in the computer system.

Although the embodiments of the invention has been described in detailwith reference to the drawings, a specific configuration is not limitedto the embodiments, and designs or the like without departing from thescope of the invention are also included.

DESCRIPTION OF THE REFERENCE SYMBOLS

-   -   11: Observation apparatus    -   41: Control device    -   4211: Area calculation unit    -   4222: Cell number calculation unit    -   4223: Density calculation unit    -   4224: Quality determination unit    -   4225: Maturity determination unit    -   43: Storage unit

The invention claimed is:
 1. An analysis method that determines amaturity degree of a cell colony during cell incubation, comprisingperforming: an information acquisition step of acquiring informationindicating a type of a cell line of which the maturity degree is to bedetermined; a calculation step of calculating, based on a captured imageof a cell during the cell incubation, an area of a target colony of thecell; a calculation step of calculating a number of cells in the targetcolony based on the area of the target colony calculated in thecalculation step; a calculation step of calculating a cell density basedon the area of the target colony calculated in the calculation step andthe number of cells calculated in the calculation step; and a step ofdetermining a maturity degree of the target colony based on the celldensity calculated in the calculation step and a calibration curve of astandard cell line that is identical to the cell line acquired in theinformation acquisition step.
 2. The analysis method according to claim1, wherein in the calculation step of calculating a number of cells, anumber of cells is calculated using the area of the target colonycalculated in the calculation step and a calibration curve of a standardcell line that is identical to the cell line acquired in the informationacquisition step.
 3. The analysis method according to claim 1, whereinin the information acquisition step of acquiring a type of a cell line,the type of a cell line input by a user is acquired, or the type of acell line is acquired according to an image analysis based on a capturedimage of the cell line.
 4. The analysis method according to claim 1,wherein the calibration curve of the step of determining a maturitydegree indicates a relationship between an area of a colony of the cellline and an area per one cell included in the colony, and in the step ofdetermining a maturity degree, the target colony is determined to bematured based on a relationship between an area per one cell included inthe target colony and a predetermined threshold value relating to anarea of one cell.
 5. The analysis method according to claim 1, whereininformation of the calibration curve for each cell line is acquired froma storage unit in which the information is stored in advance.
 6. Theanalysis method according to claim 2, wherein information of thecalibration curve for each cell line is acquired from a storage unit inwhich the information is stored in advance.
 7. The analysis methodaccording to claim 1, further comprising a step of determining whetherthe colony is good or bad based on the area of the target colonycalculated in the calculation step and a calibration curve of a standardcell line that is identical to the cell line.
 8. An observationapparatus, comprising: an imaging device that captures a cell incubatedin an incubation container; an information input interface that permitsinformation to be input indicating a type of a cell line of which amaturity degree is to be determined; and a controller unit to which eachof the imaging device and the information input interface is connected,wherein the controller is programmed to: calculate, based on an image ofthe cell during incubation captured by the imaging device, an area of atarget colony of the cell; calculate a number of cells in the targetcolony based on the calculated area of the target colony; calculate acell density based on the calculated area of the target colony and thecalculated number of cells; and determine a maturity degree of thetarget colony based on the calculated cell density and a calibrationcurve of a standard cell line that is identical to the input cell lineinformation.
 9. The observation apparatus according to claim 8, whereinthe number of cells in the target colony is calculated using the area ofthe target colony calculated by the area calculation unit and acalibration curve of a standard cell line that is identical to the inputcell line information.
 10. The observation apparatus according to claim8, wherein the information input interface acquires the type of a cellline input by a user or acquires the type of a cell line according to animage analysis based on the captured image of the cell line.
 11. Anon-transitory, computer-readable storage medium that stores a programthat causes a computer to execute: an information acquisition step ofacquiring information indicating a type of a cell line of which thematurity degree is to be determined; a calculation step of calculating,based on a captured image of a cell during incubation, an area of atarget colony of the cell; a calculation step of calculating a number ofcells in the target colony based on the area of the target colonycalculated in the calculation step; a calculation step of calculating acell density based on the area of the target colony calculated in thecalculation step and the number of cells calculated in the calculationstep; and a step of determining a maturity degree of the target colonybased on the cell density calculated in the calculation step and acalibration curve of a standard cell line that is identical to the cellline acquired in the information acquisition step.